Fermentation method for preparing a vitamin concentrate



Patented Sept. 14, 1 948 r FERMENTATION METHOD FOR PREPARING A VITAMIN CONOENTRATE Henry L. Pollard, Nelson E. Rodgers, and Reginald E. Meade, Appleton, Win, aasignors to Western Condensing Company, San Francisco, Calif., a

. corporation of California No Drawing. Application December 1, 1944,

Serial No. 566,248

3 Claims. 1

This invention relates to processes for the manufacture of biologically active materials such as vitamins by fermentation processes. More particularly, -the invention pertains to the synthesis. from lactose-containing products (in particular, lacteal material), of riboflavin and other vitamins by the action of bacteria, and, specifically. by -.the action of the bacterium Clostridium acetobutylicum.

The present application is a continuation-inpart ofthe application Serial No. 439,310, flled April 1'7, 1942, by. Henry L. Pollard, Nelson E. Rodgers and Reginald E. Meade, entitled: "Process for manufacturing a vitamin concentrate (now issued as United States Patent No. 2,369,680).

The following paragraphs describe generally a fermentation process to the improvement of which the present invention is particularly directed.

As disclosed in our copending application, we have found that the natural riboflavin content of whey or skim milk may be increased to a considerable extent by subjecting whey r skim milk under controlled conditions to the fermenting action of C'lostridium acetobutylicum. Such a fermentation synthesis of riboflavin is accompanied by the formation of neutral solvents such as ethanol, acetone and butanol, and gases such as hydrogen and carbon dioxide, which can be recovered as valuable by-products.

To prepare a lactose-containing lacteal medium such as whey or skim milk for fermentation to increase its riboflavin content, it is sterilized completely or substantially completely, as by heat treatment at about 250 F. for about to 20 minutes. In addition. the acidity of the lactea'l medium is neutralized preferably to a pH of 6 to 'l by adding an alkaline reagent such as sodium, potassium or calcium hydroxide. Calcium carbonate may be added to enhance riboflavin production. The iron content of the lacteal medium preferably is adjusted to the range of from 0.5 to less than 4.5 parts per million. In this connection it should be noted that the natural iron content of uncontaminated whey or skim milk will range from 0.10 to 0.21 part per million, while the iron content of whey contaminated, as by corrosive contact with iron containers, may reach a value above 4.5 parts per million. In the case of uncontaminated whey, the iron content may be adjusted upwardly by incorporation of suitable amounts of a soluble ferrous salt such as ferrous sulfate, while whey containing too much iron may be diluted with uncontaminated whey.

The thus prepared material, cooled to a temperature of about 100 F., is placed in a fermenting container and inoculated with C'lostridium acetobutylicum (such as described by McCoy. Peterson and Hastings in "Journal of Infectious Diseases," volume 39, page 457) preferably at a temperature about 100 F. under conditions such as will prevent the introduction of. iron and contaminating organisms. A suitable inoculum may be prepared from a stock culture by repeated transfers to a nutrient medium such as whey.

In general, fermentation of a batch of whey can continue for from twelve to forty-eight hours, or until there is no noticeable further increase in the riboflavin content.

The gases formed during fermentation can be vented from the fermenting tank as formed. The solvents formed during fermentation can be removed by fractional distillation, and after re-' moving volatile products the fermented material can be concentrated by evaporation to produce a concentrated liquor. If desired, this liquor can be further subjected to drying to produce a powdered product.

If desired, instead of separately fractionating the solvents, they can be condensed from the vapors evolved during concentration of the fermented material by evaporation to form a water-solvent mixture from which the solvents can be removed by fractional distillation.

At some point after fermentation, it is desirable to inhibit further bacterial action, as for example by heat sterilization applied as :a separate step or in conjunction with concentration by evaporation.

The product obtained by the above procedure is a concentrate which can be further reflnedor blended with various food material for human or animal consumption. By use of the process described hereinabove, the riboflavin content of whey has been increased from about 1.4 to from to micrograms per milliliter (before concentration), which corresponds to about 240 to 2800 micrograms per gram on a dried basis.

Some of the lactose is consumed in the fermenting process so that the final product contains a reduced amount of milk sugar, depending upon the extent of fermentation. The solids of the final product are in the remaining solids of the whey or skim milk employed and therefore are available as food ingredients which are used to"advantage when the produce is blended with other material, such as various milk products, bread and bakery products, poultry and animal feeds, and the like.

The present invention pertains particularly to the addition, of soluble manganese salt to a lactose-containing material to be used in the fermentation process such as that described hereinabove.

Whole milk is reputed to contain from 0.02 to 0.06 part per million of manganese. We have now found that increased riboflavin yields can be obtained by incorporating a soluble manganese salt, such as manganous sulfate, with whey or other lactose-containing lacteal material being fermented.

It is therefore an important object of the present invention to provide an improved method for synthesizing riboflavin from whey or the like by fermentation with Clostridium acetobuty1= icum.

An important object of the present invention is to provide a process of the nature indicated including the step of incorporating a soluble manganese salt with whey or other lactose-containing medium.

Other and further objects and features of the present invention will become apparent from the following description and appended claims.

In proceeding according to the present invention, the directions given hereinabove as applying to the method of the copending application are followed,.except for the variations disclosed hereinbelow.

In general, from 0.05 to 20 parts per million of manganese in the form of a soluble manganese salt may be incorporated with the lactosecontaining material such as whey being fermented. Such incorporation is most conveniently effected before the medium is sterilized.

The type of results obtained when proceeding according to the present invention are illustrated by an experiment described hereinbelow.

The basal medium used in this experiment was rennet whey supplemented with 4.8 parts per million zinc sulfate heptahydrate, parts per million par'a-aminobenzoic acid and 0.2% calcium carbonate. The activity of various amounts of manganous sulfate was tested in th presence of different concentrations of iron, added as ferrous sulfate, as indicated in the accompanying table. The various media were autoclaved in 100 milliliter volumes, inoculated with 4% of a suitable Clostrz'dium acetobutylicum starter and incubated at 100 F. for 43 hours. The riboflavin yields in duplicate cultures are shown in the following table:

It is apparent from this data that supplementin whey with manganese materially improved riboflavin production.

The above example has been given solely as illustrative of the applications of the methods of this invention to the synthesis of riboflavin from lacteal or other lactose-containing liquids. A number of experiments of this general type form the basis for the claimed methods. Isolated experiments may show minor deviations from the claimed range, but in a series of experiments consistently good average yields of riboflavin may be obtained by incorporating with from 0.05 to 20 parts per million of manganese with the lactose-containing medium in the form of a soluble manganese salt. 1

In the foregoing, particular reference has been made to the synthesis of riboflavin. It is to be understood, however, that other nutritive or vitamin factors or factors of vitamin B complex may be synthesized in addition to riboflavin.

Many details of composition and procedure may be varied within a wide range without departing from the-principles of this invention and it is therefore not our purpose to limit the scope of the patent granted on this invention otherwise than necessitated by the scope of the appended claims.

We claim as our invention:

1. In the process of manufacturing a vitamin concentrate including riboflavin by fermenting a lactose-containing lacteal material with Clostridium acetobutylicum in the presence of from 0.5 to less than 4.5 parts per million of iron, the improvement comprising incorporating with said material from 0.05 to 20 parts per million of manganese in the form of a soluble manganese salt.

2. In the process of manufacturing a vitamin concentrate including riboflavin by fermenting heat sterilized whey with Clostridium acetobutzllicum in the presence of from 0.5 to less than 4.5 parts per million of iron, the improvement which comprises incorporating with said whey prior to heat sterilization thereof from 0.15 to 30 parts per million of MnSO4.

3. In the process of manufacturing riboflavin by fermenting whey with C'lostridium acetobutylic'um, the improvement of carrying out the fermentation in the presence of from 0.5 to less than 4.5 parts per million of iron and in the presence of available manganese and regulating the manganese concentration to lie within the range of about .05 to 20 parts per million.

' HENRY L. POILARD,

NELSON E. RODGERS. REGINALD E. MEADE.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,089,562 Legg Aug. 10, 1937 2,202,161 Miner May 28, 1940 2,297,671 Yamasaki Sept. 29, 1942 2,326,425 Arzberger Aug. 10, 1943 2,369,680 Meade et al. Feb. 20, 1945 7 OTHER REFERENCES Industrial and Engineering Chem, September 1930, page 1011.

Winton, Structure and Composition of Foods, vol. III (193'!) pages 143 and 195.

Levine et al., Monograph on Systematic Bacteriology, vol. II, 1930, p e 382.

Porter, Bacterial Chemistry and Physiology, Wiley, 1946, page 628. 

